A mechanism for suppression of TNF induced endothelial dysfunction

描述(申请人提供)：在肺微血管内皮细胞中，肿瘤坏死因子(即早期~0.5小时)诱导ONOO介导的肌动蛋白硝化，导致粘连蛋白从粘连连接移位。在我们的新的活体血管损伤模型中，在肿瘤坏死因子-24.0h内，存在一个抑制血管通透性的“窗口”。我们的新的初步数据显示，在体外和/或在体内，抑制(~4.0小时)肿瘤坏死因子诱导的内皮通透性增加与核-连环蛋白移位和膜VE-钙粘附素的增加有关。我们新的初步数据表明：(1)在体外，肿瘤坏死因子-24小时诱导的屏障功能障碍可被体内糖原合成酶激酶(GSK)3a/β抑制；(2)在体外和体内，肿瘤坏死因子(TNF)可增强T细胞因子(TCF)/淋巴增强因子(LEF)/β-连环蛋白依赖的启动子活性；(3)在体内，通过抑制PKC和PKCA，这些反应被阻止。这一建议将检验这样一种范式，即肿瘤坏死因子诱导的血管通透性增加(~24.0小时后)受到抑制(~4.0小时后)，至少部分是通过PKCA诱导的对GSK3活性的抑制。GSK3？活性降低导致核连环蛋白升高。基因组连接素活性增加VE-钙粘附素的表达。VE-钙粘附素表达的增加促进了小带粘连连接，抑制(即抑制)最初(约0.5小时后)内皮细胞蛋白通透性的增加。VE-钙粘附素的增加促进了与β-连环蛋白的复合体的形成，这是连环连环蛋白持续基因组效应的关闭信号。有人认为，持续抑制GSK3的活性将继续抑制屏障功能障碍。我们将绘制一种新的范式(即体外、在体和体内)来抑制(“刹车”)肿瘤坏死因子诱导的肺损伤，这是GSK3？介导的、连环蛋白依赖的VE-钙粘附素活性增加。假设：要检验的假设是，肿瘤坏死因子诱导的肺损伤至少部分地受到粘连连接中的PKCA、GSK3、2-连环蛋白、VE-钙粘蛋白的抑制。其具体目的是在体外和/或体内确定：(1)在血管内皮细胞中，(1)肿瘤坏死因子引起PKCA介导的GSK3？抑制，从而诱导核连环蛋白移位(1-2年)；(2)肿瘤坏死因子诱导的核连环蛋白移位引起VE-钙粘蛋白的增加：(1)启动子活性；(2)RNA表达；(3)蛋白质合成(2-3年)；(3)VE-钙粘蛋白蛋白表达的增加抑制，至少部分抑制后者导致的血管屏障功能障碍的增加(3-4)。这项建议将使用大鼠肺微血管内皮细胞和体内治疗大鼠的肺组织。一系列生化、基因组、蛋白质和细胞生物学分析与肺生理学结果相结合。这种方法将在体内将细胞-分子途径转化为肺损伤的机制。

DESCRIPTION (provided by applicant): In lung microvessel endothelial cells, TNF (i.e., early ~0.5 hr) induces ONOO--mediated nitration of ¿-actin causing dislocation of ¿-catenin from adherence junctions. In our new model of in vivo vascular injury at TNF-24.0 hr, there is a "window" of suppressed vascular permeability within TNF-4.0 hr. Our new preliminary data shows, both in vitro and/or in vivo, that the suppression of (~4.0 hr) TNF-induced increased endothelial permeability is associated with nuclear ¿-catenin translocation and increases in total and membrane VE-cadherin. Our novel preliminary data demonstrates: (1) TNF-24 hr induced barrier dysfunction ex vivo is suppressed by inhibition of glycogen synthetase kinase (GSK)3a/¿ in vivo, (2) T cell factor (TCF)/ lymphoid enhancer factor (LEF)/¿-catenin dependent promoter activity is increased by TNF in vitro and in vivo, and (3) These responses to TNF are prevented by inhibition of PKC in vivo and PKCa in vitro. This proposal will test the paradigm that TNF-induced (~24.0 hr post- TNF) increase in vascular permeability is suppressed (~4.0 hr post-TNF), at least in part, by PKCa-induced inhibition of GSK3¿ activity. The decrease in GSK3¿ activity causes increased nuclear ¿-catenin. The genomic ¿-catenin-activity increases the expression of VE-cadherin. The increase in VE-cadherin expression promotes zonular-adherence junctions suppressing (i.e., braking) the initial (~0.5 hr post-TNF) increased endothelial protein permeability. The increase in VE-cadherin promotes complex formation with ¿-catenin, the off signal for continuous genomic effects of ¿-catenin. It is proposed that persistent inhibition of GSK3¿ activity will continue to suppress barrier dysfunction. We will map a novel paradigm (i.e., in vitro, in situ and in vivo) for the suppression ("braking") of TNF-induced lung injury which is GSK3¿-mediated, ¿-catenin-dependent increased VE-cadherin activity. Hypothesis: The hypothesis to be tested is TNF-induced lung injury is suppressed, at least in part by, ?PKCa ? ?GSK3¿ ? ?2-catenin ? ?VE-cadherin in adherence junctions. The Specific Aims are to determine, in vitro and/or in vivo, in endothelium that: (1) TNF causes PKCa-mediated GSK3¿-inhibition that induces nuclear ¿-catenin translocation (Years 1- 2), (2) The TNF-induced nuclear ¿-catenin translocation causes increased VE-cadherin: (I) promoter activity, (ii) RNA expression, and (iii) protein synthesis (Year 2-3), and (3) The increase in VE-cadherin protein expression suppresses, at least in part, the latter TNF-induced increase in vascular barrier dysfunction (3-4). This proposal will use rat lung microvessel endothelium and lungs isolated from rats treated in vivo. An array of biochemical, genomic, proteonomic and cell biology assays are integrated with lung physiology outcomes. This approach will translate cell-molecular pathways to the mechanisms of lung injury in vivo.